This project is centered on the mechanisms of exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. We report two subprojects this year, both related to the fact that the major exocytotic proteins are clustered. Last year, in the first project we described the creation of macroscopic raft domains in lipid membranes. We describe quantitatively the creation and evolution of phase-separated domains in a multicomponent lipid bilayer membrane. The early stages, termed the nucleation stage, and the independent growth stage, are extremely rapid (characteristic times are submillisecond and millisecond, respectively) and the system consists of nanodomains of average radius about 5 -50 nm. Next, mobility of domains becomes consequential; domain merger and fission become the dominant mechanisms of matter exchange, and line tension is the main determinant of the domain size distribution at any point in time. For sufficiently small line tension, the decrease in the entropy term that results from domain merger is larger than the decrease in boundary energy, and only nanodomains are present. For large line tension, the decrease in boundary energy dominates the unfavorable entropy of merger, and merger leads to rapid enlargement of nanodomains to radii of micrometer scale. At intermediate line tensions and within finite times, nanodomains can remain dispersed and coexist with a new global phase. The theoretical critical value of line tension needed to rapidly form large rafts is in accord with the experimental estimate from the curvatures of budding domains in giant unilamellar vesicles. This year we continue to study this mechanism in detail. The effect of an external applied lateral tension on the line tension between two domains of different[unreadable] thickness in a lipid bilayer membrane is calculated. The thick domain is treated as a liquid-ordered phase in order to model a raft in a biological membrane; the thin domain is considered a liquid-disordered phase to model the surrounding region. In our model, the monolayers elastically distort at the boundary to create a smooth rather than steplike boundary to avoid exposure of the hydrophobic interior of the thick raft to water. The energy of this distortion is described by the fundamental deformations of splay and tilt. This energy per unit length of boundary yields the line tension of the raft. Applying lateral tension alters the fundamental deformations such that line tension increases. This increase in line tension is larger when the spontaneous curvature of a raft is greater than that of the surround; if the spontaneous curvature of the raft is less than that of the surround, the increase of the line tension due to application of the lateral tension is more modest.[unreadable] The second project is experimental in nature, and uses a model for the exocytotic proteins a fusion protein expressed in fibroblasts. Organization in biological membranes spans many orders of magnitude in length scale, but limited resolution in far-field light microscopy has impeded distinction between numerous biomembrane models. One canonical example of a heterogeneously distributed membrane protein is hemagglutinin (HA) from influenza virus, which is associated with controversial cholesterol-rich lipid rafts. Using fluorescence photoactivation localization microscopy (FPALM), we are able to image distributions of tens of thousands of HA molecules with sub-diffraction resolution (30-40 nm) in live and fixed fibroblasts. HA molecules form irregular clusters on length scales from 30 nm up to many micrometers, consistent with results from electron microscopy. In live cells, the dynamics of HA molecules within clusters is observed and quantified to determine an effective diffusion coefficient. The results are interpreted in terms of several established models of biological membranes.